Erythropoietin is a glycoprotein hormone necessary for the maturation of erythroid progenitor cells into erythrocytes. It is produced in the kidney and is essential in regulating levels of red blood cells in the circulation. Conditions marked by low levels of tissue oxygen signal increases in production of erythropoietin, which in turn stimulates erythropoiesis. The erythropoietin level in the circulation is strictly regulated to ensure that red blood cells are made only in response to a long-term oxygen deficit. 70% of erythropoietin is cleared by receptor-mediated endocytosis. When erythropoietin binds to its receptor, the complex is endocytosed and degraded, thus limiting the extent of signaling. The remainder of erythropoietin is cleared through kidney filtration into the urine. As a result, erythropoietin has a relatively short serum half-life.
Naturally-occurring human erythropoietin or recombinant erythropoietin produced in mammalian cells contains three N-linked and one O-linked oligosaccharide chains. N-linked glycosylation occurs at asparagine residues located at positions 24, 38 and 83, while O-linked glycosylation occurs at a serine residue located at position 126 (Lai et al., (1986) J. Biol. Chem. 261:3116; Broudy et al., (1988) Arch. Biochem. Biophys. 265:329). The oligosaccharide chains have been shown to be modified with terminal sialic acid residues. N-linked chains typically have up to four sialic acids per chain and O-linked chains have up to two sialic acids. An erythropoietin polypeptide may therefore accommodate up to a total of 14 sialic acids. It has been shown that the carbohydrate is required for secretion of erythropoietin from cells, for increasing the solubility of erythropoietin, and for the in vivo biological activity of erythropoietin (Dube et al., (1988) J. Biol. Chem. 263:17516; DeLorme et al., (1992) Biochemistry 31:9871-9876).
Administration of recombinant human erythropoietin has been effective in treating hematopoietic disorders or deficiencies, such as, for example, different forms of anemia, including those associated with renal failure, HIV infection, blood loss and chronic disease. Erythropoietin is typically administered by intravenous injection. Since erythropoietin has a relatively short serum half-life, frequent intravenous injections are required to maintain a therapeutically effective level of erythropoietin in the circulation. Pharmaceutical compositions containing naturally-occurring or recombinant human erythropoietin are typically administered three times per week at a dose of approximately 25-100 Units/kg. This form of erythropoietin therapy, although quite effective, is very expensive and inconvenient because intravenous administration often necessitates a visit to a doctor or hospital. Currently, a hyperglycosylated recombinant human erythropoietin analogue, novel erythropoiesis stimulating protein (NESP), is available under the trademark Aranesp® (Amgen Inc., Thousand Oaks, Calif.) for treatment of anemia. Aranesp® can be administered less frequently than regular erythropoietin to obtain the same biological response.
An alternative route of administration is subcutaneous injection. This form of administration may be performed by patients at home, and is more compatible with slow-release formulations offering slower absorption from the site of administration, thus causing a sustained release effect. However, significantly lower circulation levels are achieved by subcutaneous injection and, thus, frequent injections are required to achieve desirable therapeutic effect. Furthermore, subcutaneous administration of protein drugs is generally more immunogenic than intravenous administration because the skin, as the major barrier to infection, is an immune organ that is rich in dendritic cells and has sensitive mechanisms for identifying and responding to abrasions and foreign materials. Casadevall et al. recently reported that patients receiving erythropoietin subcutaneously developed anti-erythropoietin antibodies (Casadevall et al. (2002) N Engl. J. Med. 346(7):469-75).
Accordingly, there is a need for a more efficient erythropoietin therapy that requires less frequent administrations.